高表达受体活性修饰蛋白1对血管紧张素Ⅱ和降钙素基因相关肽诱导的A10细胞降钙素受体样受体膜分布的影响  

Effect of overexpression of RAMP1 on membrane distribution of CRLR induced by angiotensinⅡ and calcitonin gene-related peptide in A10 cell line

作　　者：孙飞[1,2] 唐江琼[1] 郑元斌[1] 秦又发[1] 陈临溪[1] 秦旭平[1] 

机构地区：[1]南华大学药物药理研究所,湖南衡阳421001 [2]成都军区昆明总医院药剂科,云南昆明650032

出　　处：《中国药理学与毒理学杂志》2013年第2期209-215,共7页Chinese Journal of Pharmacology and Toxicology

基　　金：国家自然科学基金(30572192);国家自然科学基金(81173060)~~

摘　　要：目的探索受体活性修饰蛋白1(RAMP1)对血管紧张素Ⅱ(AngⅡ)和(或)降钙素基因相关肽(CGRP)诱导的降钙素受体样受体(CRLR)在血管平滑肌细胞(VSMC)的表达和分布的影响,进一步揭示CGRP抑制VSMC增殖的机制。方法 通过酶切、连接、转化等方法构建pCDNA3.1(+)-RAMP1真核表达载体并稳定转染至鼠源性血管平滑肌细胞株A10中,获得稳定高表达RAMP1的细胞系。无转染细胞、转染空载体〔pCDNA3.1(+)〕细胞和RAMP1高表达组细胞〔pCDNA3.1(+)-RAMP1〕分别用AngⅡ100 nmo.l L-1、CGRP 100 nmo.l L-1和CGRP+AngⅡ处理24 h,用MTT法检测细胞存活;逆转录PCR、Western印迹和免疫荧光法分别检测CRLR mRNA含量、蛋白表达及细胞膜分布。结果 单纯转染空质粒或RAMP1对细胞增殖无明显影响。AngⅡ处理对3种细胞存活的影响无显著差异。pCDNA3.1(+)-RAMP1细胞经CGRP处理24 h后,细胞存活明显高于其他两组细胞(P<0.05);经CGRP+AngⅡ处理,细胞存活明显低于其他两组(P<0.05)。AngⅡ,CGRP和CGRP+AngⅡ处理对3种细胞CRLR mRNA表达无明显影响,但CGRP处理使pCDNA3.1(+)-RAMP1细胞中CRLR蛋白明显高于其他两种细胞(P<0.05),而CGRP+AngⅡ处理使pCDNA3.1(+)-RAMP1细胞中CRLR蛋白明显低于其他两种细胞(P<0.05)。免疫荧光结果显示,经无血清或CGRP处理后,无转染细胞和pCDNA3.1(+)细胞中的RAMP1和CRLR主要分布在胞浆区域;经AngⅡ或CGPR+AngⅡ处理后,pCDNA3.1(+)-RAMP1细胞中RAMP1和CRLR在细胞膜上的分布多于无转染细胞和pCDNA3.1(+)细胞。结论 高表达RAMP1能增强CGRP抑制AngⅡ诱导的血管平滑肌细胞增殖,其机制可能是通过RAMP1增加CRLR的膜分布,从而增强CGRP受体对CGRP的敏感性有关。OBJECTIVE To investigate the effect of overexpression of receptor activity modifying protein 1 （RAMP1） on distribution of the calcitonin receptor like receptor （CRLR） in vascular smooth muscle cell （VSMC） in order to reveal the antiproliferative mechanism of calcitonin gene-related peptide（CGRP） for VSMC. METHODS pCDNA3.1 （ ＋ ）-RAMP1 eukaryon expression vector was successfully constructed by digestion, ligation, transform and transfected to the mouse VSMC cell line A10. After that the normal cells, pCDNA3. 1 （ ＋ ） cells and pCDNA3.1 （ ＋ ）-RAMP1 cells were treated by Ang II, CGRP and CGRP ＋Ang Ⅱ for 24 h. The proliferation of cell line A10 was determined by MTT assay while mRNA and proteins levels of CRLR and RAMP1 were determined by RT-PCR and Western blotting, respectively. The distribution of RAMP1 and CRLR in cell line A10 was observed by immunofluorescence. RESULTS Prolif- eration was not significant in three kinds of cells treated by 0.1% FBS or Angll. Proliferation in RAMP1 overexpression cell higher than in normal cells and the pCDNA3.1 （ ＋ ） cells treated by CGRP groups （ P 〈 0.05）, but lower than treated by CGRP ＋ Ang Ⅱ （ P 〈 0.05）. Cells treated with CGRP and Angll de- creased the CRLR proteins expression in RAMP1 overexpression group （P 〈0.05） while the difference of mRNA levels of CRLR in each group had no significance. However, after treated with 0. 1% FBS or CGRP, the RAMP1 and CRLR proteins were distributed into cytoplasm in normal cells and pCDNA3.1 （ ＋ ） cells, but the membrane distribution of RAMP1 and CRLR in pCDNA3.1 （ ＋ ）-RAMP1 cells were higher than that of normal and pCDNA3. 1 （ ＋ ） cells treated by Ang Ⅱ or CGRP ＋ Ang Ⅱ. CONCLUSION Overexpression of RAMP1 significantly enhances the inhibitory effect of CGRP on proliferation of VSMC induced by AngⅡ. The mechanism may be related to the increased distribution of CRLR in the membrane induced by RAMP1.

关 键 词：血管紧张素Ⅱ 降钙素基因相关肽 降钙素受体样受体 受体活性修饰蛋白1 

分 类 号：R96[医药卫生—药理学]